Electron spin-labelling of the EutC subunit in B₁₂-dependent ethanolamine ammonia-lyase reveals dynamics and a two-state conformational equilibrium in the N-terminal, signal-sequence-associated domain

Abstract

The B₁₂ (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) is a product of the ethanolamine utilisation (eut) gene cluster, that is involved in human gut microbiome homeostasis and in disease conditions caused by pathogenic strains of Salmonella and Escherichia coli. Toward elucidation of the molecular basis of EAL catalysis, and its intracellular trafficking and targeting to the Eut biomicrocompartment (BMC), we have applied electron spin-labelling and electron paramagnetic resonance spectroscopy to wild-type (wt) EAL from Salmonella typhimurium, by using the sulphydryl-specific, 4-maleimido-TEMPO (4MT) spin label. One cysteine residue per active site displays exceptional reactivity with 4MT. This site is identified as βC37 on the EutC subunit, by using 4MT-labeling of site-specific cysteine-to-alanine mutants, enzyme kinetics, and accessible surface area calculations. Electron paramagnetic resonance (EPR) spectra of 4MT-labelled wt EAL are collected over 200–265 K in frozen, polycrystalline water-only, and 1% v/v DMSO solvents. EPR simulations reveal two mobility components for each condition. Detectable spin probe reorientational motion of the two components occurs at 215 and 225 K with 1% v/v DMSO, relative to the water-only condition, consistent with formation of an aqueous-DMSO solvent mesodomain around EAL. Parallel trends in fast- and slow-reorientational correlation times and interconversion of the two populations with increasing temperature, indicate 4MT labelling of a single site (βC37). A two-state model is proposed, in which the fast and slow motional populations represent EAL-bound and free conformations of the EutC N-terminal domain. The approximately equal proportion of each state may represent a balance between EutC and EAL protein stability and efficient targeting to the BMC.

Keywords:

• Electron paramagnetic resonance
• protein conformation
• Salmonella
• signal sequence
• spin labelling

Disclosure statement

The authors declare no competing financial interest.

Additional information

Funding

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH) (Grant R01 DK054514). The Bruker E500 electron paramagnetic resonance (EPR) spectrometer was funded by the National Center for Research Resources of the NIH (Grant RR17767) and by Emory University.